The growth hormones (GH) from man and from the common domestic animals are proteins of approximately 191 amino acids, synthesized and secreted from the anterior lope of the pituitary. The growth hormone is a key hormone involved in the regulation of not only somatic growth, but also in the regulation of metabolism of proteins, carbohydrates and lipids.
During the past 40 years or more much attention has been devoted to the unravelling of the biochemical function of the growth hormones from various species. The reason for this interest in the molecular function of this protein rests upon the commercial interests from both veterinarian and medical circles. The GH gene has now been cloned and human growth hormone (hGH) and Met-hGH are currently beeing produced biosynthetically by the use of both bacteria and mammalian cell cultures.
Pharmaceutical preparations of GH tend to be unstable. Degradation products such as deamidated or sulfoxydated products and dimer or polymer forms are generated—especially in solutions of GH. Therefore, today GH is lyophilized and stored in the lyophilized form at 4° C. until it is reconstituted by the patient, before start of use.
The reconstituted preparations are preferably stored at 4° C. to minimize degradation in solution. However some degradation will take place during such storage which can be for a period of up to about 14 days. There is thus a need in the art for more stable preparations of GH.
It would also be an advantage to avoid the lyophilization step in the production of GH preparations. Lyophilization is a time consuming and costly process and also often a limiting procedure due to the capacity of the freeze drier.
The present invention is based on the surprising recognition that the above needs are fulfilled by means of a crystallization step in the production of GH.
Although readily available in quantities sufficient for crystallization, GH has so far eluded succesfull crystallization. Micro crystals, or amorphous material have been reported from a variety of sources: (Jones et al., Bio-Technology (1987) 5, 499-500; Wilhelmi et al., J.Biol.Chem. (1984) 176, 735-745; Clarkson et al., J.Mol.Biol. (1989) 208, 719-721; Bell et al., J.Biol.Chem. (1985) 260, 8520-8525 and V. L. Spitsberg, Analytical Biochemistry, 1987, 160, 489-435)
The hanging drop method is the most common method in use for this purpose. Apparently due to heterogenicity among growth hormone preparations the size and the shape of the crystals have been reported to vary significantly. The largest crystals have been reported by Jones et al. (1987). For their successfull experiments they used a mixture of polyethylene glycol 3500 and beta octyl glucoside at neutral pH. Clarkson et al. (1989) reported that the use of lower alcohols and acetone permitted the generation of crystals of 0.001 to 0.005 cubic mm with varying shapes. None of the known methods are however suitable for commercial production of GH crystals a.o. due to the fact that growth times of from several weeks up to one year are needed.
Bovine growth hormone has been formulated for veterinarian use in a mixture of divalent ions and an oil (EP 343 696). By addition of ZnCl2 to either bovine or ovine growth hormone in the presence of lipids undefined particles were produced to form a prolonged release formulation. The growth hormone was dispersed in the carrier in such a way as to trap 1 to 4 Zinc molecules per growth hormone molecule. The solutions were prepared in the presence of varying concentrations of denaturing solutes (1 to 4 M of urea) at high pH (9.5). A reproduction of this process with hGH has shown that it is not possible to produce crystals in this way.
From the literature it is well known that the presence of divalent cations during the process of crystallization permits not only excellent orientation during analysis, but also improved physical conditions for the crystallization of insulin (e.g. U.S. Pat. No. 2,174,862). Growth hormone is, however, more than three times larger than insulin and has a totally different conformation. Surprisingly the addition of cations to solutions containing hGH have now permitted the generation of stable, uniform crystals of the growth hormone in high yields. Furthermore, the time used for the formation of high quality hGH crystals is relatively short.